PROJECT SUMMARY/ABSTRACT One of the most critical events in fracture healing is reestablishment of a functional vascular network. Angiogenesis, the growth of new vessels from existing vessels, depends on both biological and mechanical cues, and growing evidence links the process of rebuilding a vascular network with the program of bone formation. Recent data show that endothelial cells (ECs) receive signaling cues from adjacent ECs as well as from multipotent stromal cells, including osteoprogenitor cells (OPCs), which reside in the ?perivascular niche?. This close spatial relationship between OPCs and ECs suggests functional codependency via cell? cell contact and/or paracrine signaling. Here we propose to elucidate the cellular and molecular basis of this effect using a novel in vivo mechanobiological model of bone repair, gene knockout, and high-resolution three-dimensional (3D) imaging modalities. CXCL12 (SDF-1) is a soluble chemokine involved in stem cell recruitment and differentiation, is expressed in skeletal stem cells, osteoblasts, and osteocytes, and is upregulated in response to skeletal injury and mechanical loading. Blood vessels express CXCR4 and endothelial cells co-localize with CXCL12-expressing cells at the injury site, suggesting that CXCL12 may play a critical role in angiogenesis during bone repair. The overall goal of this project is to elucidate the role of CXCL12/CXCR4 signaling in osteo-angio coupling in bone. Our central hypothesis is that OPCs locally regulate Type H vessels through CXCL12 signaling, and that the mechanical strain environment is communicated to the local vasculature through modulation of CXCL12 expression. Our Aims are (1) To demonstrate functional codependency between OPCs and Type H vessels during bone repair, (2) To determine the influence of CXCL12/CXCR4 signaling on OPC-EC coupling. (3) To perform unbiased profiling of CXCL12+ cells during bone repair with and without mechanical stimulation. Results from these studies will advance our fundamental understanding of mechanisms regulating osteogenesis-angiogenesis coupling during bone repair and may reveal a key signaling pathway regulating angiogenesis in bone. Our findings would directly impact pro-angiogenic biologics and current indications for autologous stem cell injections to treat diminished angiogenesis, delayed healing, and nonunion.